Diabetes is a chronic disease that afflicts 200 millions people worldwide. Type 1 diabetes results from autoimmune destruction of beta cells, while type 2 diabetes is caused by a combination of insulin resistance and inadequate insulin secretion. Thus, in both type 1 and type 2 diabetes, the functional beta cell mass is not sufficient to control glycemia.
The mature pancreas contains two types of tissue: exocrine tissue composed of acinar cells that produce enzymes (e.g., carboxypeptidase-A) secreted via the pancreatic ducts into the intestine and endocrine islets composed of cells that produce hormones such as insulin (beta cells), glucagon (alpha cells) somatostatin (delta cells) and pancreatic polypeptide (PP cells). Over the past decades research in the beta cell field profited from the establishment of insulin-secreting cell lines, such as RIN and INS1 cells derived from x-ray induced rat insulinoma (Asfari et al., 1992; Gazdar et al., 1980), HIT cells generated by transformation of hamster islet cells by SV40 (Santerre et al., 1981) and beta TC and Min6 cells derived from transgenic mice expressing SV40 T antigen under the control of the insulin promoter (Efrat et al., 1995; Efrat et al., 1993; Efrat et al., 1988; Hanahan, 1985; Knaack et al., 1994; Miyazaki et al., 1990). Such cell lines were useful for a better understanding of beta cell biology and could be used for drug screening.
Generation of pancreatic beta cells in large amount represents an important objective, because such beta cells could be used for cell therapy of diabetes. In addition, such pancreatic beta cells would also be useful for screening of new drugs that can modulate beta cell function. To this end, different approaches have been previously developed to generate pancreatic beta cells in large amount.
The first one consisted in using as starting material immature embryonic stem cells (ES cells) to produce mouse or human beta cells. The major advantage is that ES cells self-renew indefinitely in culture, and have the capacity to differentiate to multiple cell types, and thus to pancreatic beta cells. While quite a large amount of publications appeared during the past years on beta cells production from ES cells (Assady et al., 2001; Blyszczuk et al., 2003; Brolen et al., 2005; Hori et al., 2002; Lumelsky et al., 2001; Soria et al., 2000), other publications described pitfalls in such works, questioned the interpretations and demonstrated that reproducible protocols were not yet available to produce beta cells from ES cells (Hansson et al., 2004; Rajagopal et al., 2003).
Thus, at that point, functional beta cells have not yet been generated in large quantities from ES cells with the exception of one recent publication where beta cells developed from hES cells (D'Amour et al., 2006). However, such cells did not secrete insulin upon glucose stimulation.
A second approach was to derivate beta cell lines from beta cell tumours derived from transgenic mice expressing SV40 T antigen under the control of the insulin promoter (Efrat et al., 1995; Efrat et al., 1993; Efrat et al., 1988; Hanahan, 1985; Knaack et al., 1994; Miyazaki et al., 1990). These cell lines have been extremely useful for detailed study of rodent beta cells. However, as many differences exist between rodent and human beta cells, these beta cells cannot be used for human diagnosis or therapy. Also, since these beta cell lines were obtained by gene transfer in fertilized eggs, such a method is restricted to animal models without any possible transfer to human.
A third approach has been carried out by Ravassard et al. (2011). These authors described the obtention of a stable functional human beta cell line, designated EndoC-βH1, with glucose-inducible insulin secretion by using human foetal pancreases cells transduced with lentiviral vectors expressing SV40LT under the control of the insulin promoter. In this approach, the transduced pancreases cells were grafted into SCID mice so that they could develop into pancreatic tissue. The human beta cells differentiated, expressed SV40LT concomitantly with insulin, proliferated, and formed insulinomas. These insulinomas were next transduced with a lentiviral vector that expressed hTERT, and the hTERT-transduced insulinomas cells were then regrafted into other SCID mice in order to further amplify the proliferating beta cells. After removing the transplanted tissue from these SCID mice, cells were dissociated and then expanded in culture as cell lines. The resulting EndoC-βH1 cells contained 0.48 μg of insulin per million cells, were stable at least for 80 passages, and expressed many specific beta cell markers, without any substantial expression of markers of other pancreatic cell types. EndoC-βH1 cells secrete insulin in response to glucose stimulation, and insulin secretion is enhanced by known secretagogues, such as exendin-4, glibenclamide, and leucine. Finally, transplantation of EndoC-βH1 cells into mice with chemically-induced DM normalizes their glycaemia.
However it is of interest to obtain beta cells generated from a more mature post natal pancreas. In particular, embryonic pancreases can only be obtained after termination of pregnancy, which may raise ethical or legal questions in a number of countries. Thus it would be preferable to use non-embryonic material for generating human pancreatic beta cell lines.
Several attempts have been made to generate human beta cell lines from many human pancreatic sources, such as adult islets or insulinomas. However, insulin production by these cells was extremely low or these cells were capable of producing insulin only over a few passages (de la Tour et al., 2001; Demeterco et al., 2002; Gueli et al., 1987; Ju et al., 1998; Levine et al., 1995; Soldevila et al., 1991). In 2005, Narushima et al (Narushima et al., 2005) reported that they successfully established a functional human beta cell line (NAKT-15) from freshly isolated adult pancreatic islet cells transduced with a Moloney retrovirus expressing the SV40 Large T antigen. However, serious doubts have been raised regarding this work. In particular, gene transfer by such retroviral vectors occurs only in cells actively replicating at the time of infection (Miller D G et al, 1990), while adult pancreatic cells are very poorly mitotic (see Chen et al, 2011 and Kohler et al, 2010). It is thus likely that the NAKT-15 cell line is not a functional human beta cell line, as claimed, and that the method described in Narushima et al. does not enable the skilled person to obtain such a functional human beta cell line. Indeed, the work of Narushima et al. has not been reproduced, either by the authors or by other laboratories, since the original publication.
Meier J J. et al, (2008) showed that human post-natal beta-cells mass increase is due to the active replication of differentiated beta-cells and not to remaining progenitor cells. Yet these proliferating differentiated cells are incompetent to produce functional beta cell lines. Ravassard et al. (2011) attempted to generate a functional human beta cell line from adult human islets transduced by lentiviral vectors. The transduced cells survived after their transplantation into immuno-incompetent mice, but did not then develop into insulinomas. These observations were consistent with those obtained in another communication reporting that adult beta cells are refractory to transformation using multiple oncogenic mutants (Gidekel Friedlander et al, 2009).
As of today, the formation of insulinoma from human non-foetal cells has never been observed, and it has been considered impossible to obtain human beta cell lines from non-foetal pancreatic material (Ravassard et al., 2011).
Thus, there is still a need for a reliable and reproducible method for developing a functional human beta cell line from non-foetal pancreatic material.